Project Summary Half of all brain tumors are gliomas, of which glioblastoma multiforme (GBM) is the most deadly. With treatment, GBM mean life expectancy is 14.6 months. The oncogene and enzyme isocitrate dehydrogenase 1 (IDH1) catalyzes the oxidative decarboxylation of isocitrate (ISC) to ?-ketoglutarate (2-KG) in normal tissues. However in GBM, IDH1 catalyzes the reverse reaction, reductive carboxylation of 2-KG, and is upregulated at the protein level. Post translational modification (PTM) is a common way of modifying and regulating enzyme activity, and may regulate IDH1 activity in GBM, since 27 different amino acids are reportedly modified in IDH1. However, the function and combinations of these modifications has not been reported. To understand the significance of PTMs in regulating IDH1 activity, it is necessary to first measure the precise post-translationally modified variants (proteoforms) of IDH1 which exist in glioma and normal cells (Aim 1), and then to functionally characterize and genetically manipulate these variants (Aims 2 and 3, respectively). In Aim 1, the technology of top-down proteomics will be used to measure the proteoforms of IDH1 which exist in normal and glioma cell lines, as well as in resected human glioma tissue samples. This will be achieved through measurement of intact protein isoforms. In Aim 2, metabolomics and chemical labeling methods will be used to measure the catalytic activity and chemical reactivity of IDH1 proteoforms, providing insight into IDH1 proteoform function. In Aim 3, untargeted shotgun proteomics will be used to identify kinases, acetylases, phosphatases, and other PTM-installing enzymes that correlate with changes in IDH1 proteoform. The effect of these enzymes on IDH1 proteoform will then be directly tested by shRNA knockdown and overexpression in order to identify enzymes involved in proteoform synthesis.